Phosphate-responsive nanocomposite hydrogel laden with umbilical cord blood exosomes and nanosilver accelerates diabetic and infectious wound healing

2.1. Separation and identification of exosomes

UCB samples were collected from healthy neonates following the acquisition of informed consent from the mothers and approval from the Hubei University of Medicine Ethics Committee (2024KS49). As a first step, the plasma was separated from the UCB samples by centrifugation at 300×g for 10 mins at 4°C. Then, the plasma underwent sequential centrifugation at 300×g for 10 mins, 2000×g for 10 mins, and 8000×g for 35 mins, all conducted at 4°C. Subsequently, the resultant supernatant was further centrifuged at 100,000×g for 70 mins at 4°C to ensure complete removal of the liquid phase. The resulting precipitate was resuspended in phosphate-buffered saline (PBS). The protein concentration of the exosome was quantified by a bicinchoninic acid (BCA) assay kit (Beyotime, China). The UCB-exos were either utilized for experimental procedures or stored at -80°C for future use.

Transmission electron microscopy (TEM) was used to investigate the morphology of the exosome, while its size was determined with a Zeta sizer (ZS90). Surface markers of the exosome were identified using Western blot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the exosome protein, then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated overnight with antibodies against CD9 (1:1000, Proteintech, USA), CD63 (1:1000, Proteintech), CD81 (1:1000, Proteintech), TSG101 (1:1000, Proteintech), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Proteintech). Following this, it was incubated with secondary antibodies for 1h. Chemiluminescence gel imaging (Bio-Rad) and enhanced chemiluminescence detection kits (Beyotime, China) were used to visualize and analyze protein bands.

2.2. Uptake of UCB-exos

To label exosomes, the PKH67 kit (Sigma) was employed as per the manufacturer’s guidelines. The labeled exosomes were then incubated with the cells at 37°C in a 5% CO₂ environment for 24 hrs. After incubation, the cells were fixed with 4% paraformaldehyde for 15 mins, then stained with 4’,6-diamidino-2-phenylindole (DAPI) (Beyotime). Fluorescence signals were analyzed with a Leica DMI6000B microscope.

2.3. Cell proliferation, migration, and tube formation assays

Human umbilical vein endothelial cells (HUVEC) were procured from the China Center for Type Culture Collection (Wuhan, China). Minimum essential medium alpha (MEM-alpha) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin solution (Beyotime) was used to culture these cells. Dulbecco’s modified eagle medium (DMEM) (Gibco) supplemented with 10% FBS was used to culture human skin keratinocytes HaCaT (FuHeng Biology, Shanghai, China). The cells were incubated in the incubator at 37°C, 5% CO₂. The cells were divided into three groups: 1) Negative control (NC) group: culture medium; 2) High Glucose (HG) group: 30 mM Glucose; and 3) HG+exos group: 30 mM Glucose+UCB-exos for the cell proliferation, migration, and tube formation experiments.

2.4. RNA-sequencing

RNAs were extracted from HUVECs exposed to high glucose, with or without exosomes, and sequenced by Aksomics (Shanghai, China) using Illumina. The differential genes were then analyzed for biological process (BP), cell components (CC), molecular function (MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

2.5. Analysis of the cell cycle by flow cytometry

HUVECs (1×10⁶) from various treated groups were collected and stained by a cell cycle analysis kit (Beyotime) following the manufacturer’s protocol, then the flow cytometry was conducted with a FACScan flow cytometer (BD Biosciences) and analysis using ModFit software.

2.6. Preparation of PAA-CaP@Ag Nps@exo hydrogels

First, Ag nanoparticles (AgNps) were synthesized by dissolving 90 mL of a 0.15 mol/L AgNO₃ solution in a 600 mL 4.5 mmol/L Na₂HPO₄ solution. The resulting precipitate was subsequently suspended in deionized water and subjected to hydrothermal heating at 160°C for a period of two hrs and dried into a powdered form. After that, 8 mg of AgNps were added into 4 mL of phosphate buffer and dispersed using an ultrasonicator to create solution A. Then, combined 10 mL of 7.22 mg/mL PAA, 10 mL of 0.1 mol/L CaCl₂ solution, and 60 μg UCB-exos at room temperature to form solution B. Finally, solution A was added to solution B at the rate of 1 mL/min and stirred at 500 rpm for 30 min until the mixture transitioned from clarified to turbid, resulting in the formation of the PAA-CaP@AgNps@exo hydrogels.

2.7. Characterization of PAA-CaP@Ag Nps@exo hydrogels

The morphology of the freeze-dried hydrogels was studied through scanning electron microscopy (SEM). A NanoSEM 200 electron microscope operating at a 10 kV acceleration voltage was used to observe the samples. A Fourier Transform Infrared Spectrometer (FTIR) (Nicolet iS50, Thermo-Fisher, USA) was used to determine the chemical composition of the hydrogels. A Nano-Zs Malvern nano analyzer was used to detect the particle size. The structural characterization of the hydrogels was conducted using X-ray diffraction (XRD) analysis with a RINT-2000 Rigaku diffractometer (Rigaku, Tokyo, Japan). The quantitative elemental analysis and spatial distribution of elements within the hydrogels were performed using energy-dispersive X-ray spectroscopy (EDX). Elemental data and EDX mapping analyses were conducted with a Zeiss Ultra Plus SEM (ZEISS, Germany).

2.8. The release kinetics of exosome from PAA-CaP@AgNPs@exo hydrogels

In vitro release kinetics of UCB-exo from the hydrogels were evaluated by slightly modifying the previously reported method [21]. Briefly, the PAA-CaP@exo and PAA-CaP@AgNPs@ exo nanoparticle hydrogels were incubated in sterile PBS at 37°C. The protein content of UCB-exos was quantified using the microBCA protein assay (System Biosciences) following the manufacturer’s protocol.

2.9. The release kinetics of Ag ⁺ and Ca^{2 +} from PAA-CaP@AgNPs@exo under phosphate response

Equal quantities of PAA-CaP@AgNps@exo dry hydrogels were immersed in PBS solutions with molarities of 0, 0.01, and 0.05 M. The solutions were subsequently extracted at 37°C at 0, 6, 12, 24, and 48 hrs. Following extraction, 10% nitric acid (HNO₃) was added to the solutions, and they were then filtered through a needle filter with 0.22 μm. Inductively coupled plasma optical emission spectrometry was used to detect the concentrations of Ag⁺ and Ca²⁺.

2.10. In vitro antimicrobial activity

The disc diffusion method was used to evaluate the antimicrobial efficacy of hydrogels against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The hydrogels were applied to agar plates inoculated with S. aureus and E. coli and then incubated at 37°C. After 24 hrs, the diameters of the inhibition zones were measured and recorded.

2.11. Diabetic wound and diabetic wound infection models

Sprague-Dawley (SD) rats (Male, 8 weeks old, 200-220 g) were housed in an environment maintained at constant temperature and humidity. A dose of 65 mg/kg of streptozotocin (STZ) was administered intraperitoneally to the rats. Two weeks post-injection, blood glucose levels were assessed using a blood glucose monitor. Rats exhibiting blood glucose concentrations exceeding 16.67 mmol/L were classified as diabetic. To develop a diabetic wound model, diabetic rats were anesthetized via intraperitoneal administration of Nembutal at a dosage of 35 mg/kg (Sigma, USA). Following the induction of anesthesia, the dorsal skin of each rat was shaved and sterilized using 75% ethyl alcohol. Subsequently, a full thickness square wound measuring 1.5 cm on each side was surgically created on the dorsum of each mouse. Then, a total of 15 rats were randomly divided into 5 groups of three: Blank control group (BC); Aquacell ® wound dressing was used for a positive control group (PC); PAA-CaP@AgNps; PAA-CaP@exo group; and PAA-CaP@AgNps@exo group. A wound area measurement was performed using Image J on days 0, 4, 8, 12, and 16. The Hubei University of Medicine Animal Care and Use Committee approved this experiment (2024-55). The diabetic wound infection model was developed by anesthetizing diabetic rats and making wounds in the same manner as the diabetic wound model for rats. Then, S. aureus and E. coli bacteria were inoculated into the wounds at a 1:1 ratio. Three days after infection, six rats were randomly divided into two groups of three per group: PBS (control) group and PAA-CaP@Ag Nps@exo group. Digital photographs were captured on days 0, 4, 8, 12, and 16, and the wound area was quantified utilizing ImageJ software. This study received approval from the Hubei University Animal Care and Use Committee (approval number: 20240034). The rate of wound closure was calculated by assessing the wound area in each group through the delineation of wound boundaries with software (ImageJ). According to the following formula, wound closure rates (%) were calculated as follows: Wound closure rate (%) = [(S (0 day) - S (n day)/S (0 day)] × 100%, where S represents the wound area.

2.12. Histology, immunohistochemistry, and immunofluorescence analysis

A specimen of fully thickened traumatic tissue, measuring approximately 3 mm in size, was obtained from the peripheral margin of the entire wound and promptly fixed in a 4% paraformaldehyde solution. After 24 hrs, the skin wounds underwent dehydration overnight and were subsequently embedded in paraffin. Tissue sections were mounted on glass slides for histological examination. Hematoxylin and eosin (H&E) staining, along with Masson’s trichrome staining, were conducted to visualize pathological alterations within the tissue. Furthermore, paraffin-embedded skin wound sections were sliced and subjected to immunohistochemical analysis for CD31 (Proteintech, 28083-1-AP, 1:4000), Ki67 (Proteintech, 28074-1-AP, 1:2000), CD206 (Proteintech, 18704-1-AP, 1:1000), CK10 (Proteintech, 16855-1-AP, 1:200), Collagen I (Proteintech, 67288-1-Ig, 1:3000), and Collagen Ⅲ (Proteintech, 22734-1-AP, 1:1000). Photographs were taken using a microscope and digital camera. To assess the presence of pro-inflammatory cytokines at the wound site, immunofluorescence analysis was conducted on wound sections on the seventh day post-injury. We incubated the tissue sections with anti-IL-1 rabbit polyclonal antibody (pAb) (Servicebio, 1:500) and anti-tumor necrosis factor (TNF)-αrabbit pAb (Servicebio, 1:200), followed by incubation with a goat anti-rabbit IgG Alexa Fluor® 594-conjugated secondary antibody (Abcam, 1:500) and DAPI for nuclear staining. Stained sections were imaged using a fluorescent microscope, and the percentage of positively stained cells in each experimental group was calculated using Image-Pro Plus software.

2.13. Statistical analysis

Statistical analyses were conducted employing GraphPad Prism software. Data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to determine statistically significant differences between groups, with p-values less than 0.05 regarded as significant.

3.1. Exosomes characterization

UCB-exos were isolated via ultracentrifugation. Different characterization techniques such as western blotting, nanoparticle tracking analysis (NTA), and TEM analysis were used to identify the harvested UCB-exos. The Western blotting analysis confirmed the presence of CD9, CD63, CD81, and TSG101 in the isolated UCB-exos compared with the peripheral blood mononuclear cells (PBMCs) (Figure 1a). NTA suggested that the size distribution of UCB-exos displayed a single peak at approximately 100 nm (Figure 1b). As shown by TEM, UCB-exos have a typical “saucer-like” structure (Figure 1c). Together, these results confirmed the extraction of UCB-exos had been successful. Exosomes labeled with PKH67 were co-cultured with HUVEC in order to further clarify whether UCB-exos are capable of being endocytosed into cells. Our observations indicate that PKH67-labeled exosomes, identifiable by their green fluorescence, were predominantly localized in the perinuclear region of human umbilical vein endothelial cells (HUVECs), as illustrated in Figure 1(d). This finding substantiates the internalization of exosomes by endothelial cells.

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Figure 1.

UCB exosome identification. (a) Exosome-specific markers such as Tetraspanin 9 (CD9), Lysosomal membrane-associated glycoprotein 3 (CD63), CD81, and Tumor susceptibility antigen 101 (TSG101) were detected by Western blot; (b) Nanoparticle tracking analysis (NTA) for determining the size of exosome particles (30 to 150 nm); (c) Morphological observation of exosomes under transmission electron microscopy; and (d) Internalization of PKH67-labelled exosomes by cells was observed using laser confocal microscopy. PBMCs: peripheral blood mononuclear, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, Exo: exosomes, DAPI: 4’,6’-Diamidino-2-phenylindole, Blue is the nucleus, Green is the exosomes.

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3.1.1. The influence of UCB-exos on keratinocytes and endothelial cells under high glucose conditions

Keratinocytes and endothelial cells play a vital role in wound healing [22]. To examine the effects of UCB-exos on keratinocytes and endothelial cells function under high glucose. HaCaT cells and HUVECs were treated with LG, HG, and HG+exos, respectively. The proliferation of HaCaT cells and HUVECs was detected by RTCA assay. We found that high glucose concentration inhibits the proliferation of cells. At the same time, UCB-exos promoted cell proliferation under high glucose conditions (Figure 2a). The UCB-exos treatment significantly enhanced cell migration under high glucose conditions compared to the HG group, as demonstrated by the scratch wound healing assay (Figure 2b) and the transwell migration assay (Figure 2c). Then, the impact of UCB-exos on the angiogenic activities of endothelial cells was assessed. The matrigel tube formation assay serves as an in vitro model for studying angiogenesis. As shown in Figure 2(d), HUVECs treated with UCB-exos produced more capillary-like structures in high glucose conditions compared with the HG group. These results demonstrate the potential of UCB-exos in protecting the biological functions of keratinocytes and HUVECs in vitro under high glucose conditions.

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Figure 2.

The influence of umbilical cord blood exosomes (UCB-exos) on keratinocytes and endothelial cells under high glucose conditions. (a) Detection of the proliferation of human skin keratinocytes (HaCaT) cells and Human umbilical vein endothelial cells (HUVEC) with UCB-exos by real-time cell analysis (RTCA) assay; (b) Determination of the cell migration (the Migrated cells were stained purple by crystal violet.) of HaCaT cells and HUVECs with UCB-exos by scratch wound healing assay; (c) Detection of the cell migration of HaCaT cells and HUVECs with UCB-exos by transwell assay; (d) Assessment of the impact of UCB-exos on angiogenic activities of HUVECs by tube formation assay (n=3, mean±SD, *p < 0.05, **p < 0.01, ***p < 0.001). The black dots represent the experimental results from independent repetitions of the experiment. The Migrated cells were stained purple by crystal violet. HG: High glucose, NC: Negative control.

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3.1.2. Mechanism of UCB-exos regulating the biological functions of endothelial cells

To elucidate the underlying mechanisms by which UCB-exos influence the biological functions of endothelial cells under hyperglycemic conditions, transcriptome sequencing on endothelial cells treated with UCB-exos in a high glucose environment was performed. The results were compared to those of a control group subjected solely to high glucose conditions, and the findings are presented in the accompanying heatmap (Figure 3a). A total of 934 significantly different genes were obtained, including 409 differentially up-regulated genes and 525 differentially downregulated genes. The distribution of differential genes was generated to show in the volcanic map (Figure 3b). Moreover, GO function enrichment analyses were also carried out. The results are shown in Figures 3(c-e). According to the enrichment results of GO’s molecular function, biological process, and cell components, these genes are associated with biological processes such as the regulation of protein binding, response to stimulus, and intracellular anatomical structure. The enrichment results of the KEGG pathway showed that it mainly regulated the cell cycle signaling pathway (Figure 3f). Flow cytometry was used to determine the distribution of cells during the G1, S, and G2 phases of the cell cycle. The result showed that the cell cycle was halted at the transition from G1 to S-phase under glucose conditions, and this was reversed by being treated with UCB-exos (Figure 3g). It shows that UCB-exos have the potential to influence the bioactivity of endothelial cells in hyperglycemic conditions via multiple mechanisms.

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Figure 3.

The underlying mechanism of the effect of umbilical cord blood exosomes (UCB-exos) on the biological functions of endothelial cells under high glucose. (a) The heatmap of Human umbilical vein endothelial cells (HUVECs) and HUVECs treated with UCB-exos under high glucose by transcriptome sequencing; (b) The volcanic map of HUVECs and HUVECs treated with UCB-exos under high glucose by transcriptome sequencing; (c-e) Gene Ontology (GO) function enrichment analysis molecular function (MF), biological process (BP), and cell components (CC); (f) Transcriptome sequencing enrichment results of the Kyoto encyclopedia of genes and genomes (KEGG) pathway; (g) Analysis of cell cycle stages of HUVECs, and HUVECs treated with UCB-exos under high glucose by flow cytometry (n=3, mean±SD, *p < 0.05, **p < 0.01, ***p < 0.001). The pink peak represent G1 phase of the cell cycle and the blue peak represent G2 phase of the cell cycle. HG: High glucose, NC: Negative control

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3.2. Nanocomposites hydrogels characterization

To characterize the morphological structure of AgNPs, the spherical silver nanoparticles coated with phosphate were determined through TEM analysis, whereas the average particle diameter was determined through dynamic light scattering (DLS), which showed a diameter of approximately 40.2±13.6 nm (Figure 4a,b). SEM was used to investigate the hydrogel’s internal structure. The hydrogel shows an interconnected porous network structure, and the distribution of AgNps and UCB-exos into hydrogel is shown to be quite constant and homogeneous (Figure 4c). The composition and elemental constituents of the hydrogel were further examined utilizing EDS. The findings, presented in Figure 4(d-f), demonstrate that Ag nanoparticles were successfully incorporated into the hydrogel through the biomimetic mineralization technique. The XRD measurements were performed to examine the phase analysis of AgNps and PAA-CaP@AgNps (Figure 4g). According to the results, the characteristic peaks of AgNps disappeared after embedding in the PAA-CaP hydrogel, which also indicated that the AgNps had been immobilized in the PAA-CaP hydrogel. To assess and check the formation of cross-linked hydrogel networks with AgNPs and UCB-exos, the samples were analyzed by FTIR. In the FTIR spectra shown in Figure 4(h), the presence of characteristic peaks of hydrogen phosphate at 1130 cm^-1 and 987 cm^-1, shows the successful loading of AgNps. The absorption peaks near 1700 cm^-1 were obviously enhanced, indicating the successful loading of the exosome, and the change of the COO^- characteristic peaks near 1554 cm^-1 and 1454 cm^-1 speculated that the exosome had an effect on the COO^- coordination. Comparing PAA-CaP@AgNps with PAA-CaP@AgNps@exo, it can be found that the carboxylate characteristic peaks near 1620 cm^-1 and 1400 cm^-1 were significantly weakened, suggesting that the exosome was successfully loaded to PAA-CaP@AgNps. To assess the release of the exosomes from the exosome-loaded hydrogel, the hydrogel was immersed in PBS solution. Based on the MicroBCA protein assay, gradual exosome release was observed over a period of 16 days from the hydrogel (Figure 4i). These results confirmed that the exosomes could gradually be released from the hydrogel and exosomes were successfully loaded into the hydrogel.

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Figure 4.

Characterization of the nanocomposites hydrogels. (a) Scanning electron microscopy (SEM) analysis of the morphological structure of AgNPs; (b) Determination of the size of AgNPs by dynamic light scattering (DLS) measurement; (c) SEM analysis for the morphological structure of PAA-Cap, PAA-Cap@AgNps, PAA-Cap@exo, and PAA-CaP@AgNps@exo hydrogels; (d-f) Analysis of the composition and elemental constituents of PAA-CaP@AgNps@exo hydrogel by energy dispersive spectrometer (EDS); (g) Analysis of the phase of AgNps and PAA-CaP@AgNps by X-ray diffraction (XRD) measurements; (h) PAA-Cap@ AgNps@exo hydrogel was analyzed by Fourier transform infrared spectrometer (FTIR); (i) Detecting exosome release from PAA-Cap@AgNps@exo hydrogels in phosphate buffered saline (PBS) solution by micro Bicinchonic acid assay (BCA) protein assay.

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3.2.1. Phosphate response degradation of the PAA-CaP@AgNps@exo hydrogel

Due to the precipitation/dissolution equilibrium of CaPs in phosphate buffer solution, the dissolution of CaPs as cross-linking points may lead to degradation of PAA-CaPs@AgNps@exo hydrogel. To investigate the effect of different phosphate concentrations on hydrogel degradation, Ca²⁺, and Ag⁺ release from the hydrogels by treating them with different concentrations of PBS at different times were detected, respectively (Figure 5a). The findings indicated that elevated phosphate concentrations could diminish Ca²⁺ release, with Ca²⁺ release initially increasing during the first six hours and stabilizing after twelve hours (Figure 5b). In contrast, Ag⁺ exhibited an inverse response to phosphate concentration, as the high levels of PO₄^3- in PBS interacted with Ag⁺ to form a precipitate (Ag₃PO₄), thereby enhancing Ag⁺ release. The release of Ag⁺ demonstrated a positive correlation with time, achieving its maximum release after six hours. The reduced release of Ag⁺ ions at a concentration of 0.01 M PBS significantly mitigated the accumulation of heavy metals. In contrast, the control group exhibited the highest Ag⁺ release, likely attributable to the swelling of the mineralized hydrogel, as depicted in Figure 5(c). These findings suggest that the hydrogel synthesized via the bionano-mineralization method facilitates phosphate-responsive degradation and the subsequent release of encapsulated nanoparticles.

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Figure 5.

Phosphate response release Ca2⁺ and Ag⁺ from the PAA-CaP@AgNps@exo hydergel. (a) Degradation of the hydrogel with different concentrations of phosphate buffered saline (PBS) for different times; (b) Determination of the release of Ca²⁺ from the hydrogels by treating with different concentrations of PBS for different periods of time by Inductively coupled plasma optical emission spectroscopy (ICP-OES); (c) Determination of the release of Ag⁺ from the hydrogels by treating with different concentrations of PBS for different periods of time by ICP-OES.

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3.2.2. The antimicrobial properties of PAA-CaP@AgNps@exo hydrogel in vitro

The ring-of-inhibition assay compared the antimicrobial activities of AgNps, PAA-CaPs@AgNps, and PAA-CaPs@AgNps@exo hydrogels. The results indicated that the average diameters of the inhibitory rings for these three were 12, 15, and 23 mm for S. aureus, and 6, 7, and 11 mm for E. coli respectively, as shown in Figures. 6(a) and 6(b). These results showed that PAA-CaPs@AgNps@exo hydrogel was significantly more effective than the other two in inhibiting E. coli and S. aureus. The antimicrobial effect of PAA-CaPs@AgNps@exo hydrogel was further quantitatively evaluated by plate counting method, and the results showed that the inhibitory effect of the PAA-CaPs@AgNps@exo hydrogel was 100% against both E. coli and S. aureus, respectively (Figure 6c,d). It showed that the PAA-CaPs@AgNps@exo hydrogel had good bactericidal ability in vitro.

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Figure 6.

The antimicrobial properties of hydrogels in vitro. (a) Detection of the antimicrobial activities of silver nanoparticles (AgNps), PAA-CaPs@AgNps, and PAA-CaPs@AgNps@exo hydrogels on S. aureus and E. coli by the ring-of-inhibition assay; (b) Statistical results of the inhibitory rings of the AgNps, PAA-CaPs@AgNps, and PAA-CaPs@AgNps@exo hydrogels on S. Aureus and E. coli; (c) The antimicrobial effect of PAA-CaPs@AgNps@exo hydrogel evaluated by plate counting method; (d) Quantitative results of the inhibitory effect of PAA-CaPs@AgNps@exo hydrogel on E. coli and S. Aureus (n=3, mean±standard deviation (SD), *p < 0.05, **p < 0.01, ***p < 0.001). The black circles represent the experimental results from independent repetitions of the experiment.

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3.2.3. PAA-CaP@AgNps@exo hydrogel accelerates diabetic wound healing

To test whether the PAA-CaP@AgNps@exo hydrogel could accelerate diabetic wound healing, a rat model of diabetic wounds was established as shown in Figure 7(a). Wound images were captured on days 0, 4, 8, 12, and 16, as illustrated in Figure 7(b). The residual wound area was subsequently calculated for all groups at different time points. The results of the study showed that PAA-CaP@AgNps@exo hydrogel demonstrated a significantly faster wound healing rate than any of the other groups (Figure 7c). The dynamic recovery process of wound healing across different groups is shown in Figure 7(d). Skin tissue samples surrounding the wounds were subjected to HE and Masson staining. The results indicated complete epithelial formation in the PAA-CaP@AgNps@exo group. In addition, there was a substantial migration of fibroblasts, a significant increase in blood vessel formation, and a significantly higher incidence of hair follicle formation than in the other groups (Figure 7e). According to these results, PAA-CaP@AgNps@exo hydrogel accelerates diabetic wound healing.

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Figure 7.

The antimicrobial properties of hydrogels in vitro. (a) Detection of the antimicrobial activities of AgNps, PAA-CaPs@AgNps, and PAA-CaPs@AgNps@exo hydrogels on S. aureus and E. coli by the ring-of-inhibition assay; (b) Statistical results of the inhibitory rings of the AgNps, PAA-CaPs@AgNps, and PAA-CaPs@AgNps@exo hydrogels on S. Aureus and E. coli; (c) The antimicrobial effect of PAA-CaPs@AgNps@exo hydrogel evaluated by plate counting method; (d) Quantitative results of the inhibitory effect of PAA-CaPs@AgNps@exo hydrogel on E. coli and S. Aureus. (n=3, mean±SD, *p < 0.05, **p < 0.01, ***p < 0.001). (e) Histological examination the skin tissue of different groups by Hematoxylin and eosin (HE) staining and Masson’s trichrome (Masson) staining (50x). BC: Blank control, PC: Positive control.

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3.2.3. PAA-CaP@AgNps@exo hydrogel promotes angiogenesis, cell proliferation, anti-inflammation, re-epithelialization, and collagen deposition

To elucidate the mechanism of action of the PAA-CaP@AgNps@exo hydrogel in promoting wound healing, immunohistochemistry was employed to assess the expression of CD31, Ki67, and CD206 in the wound skin tissues of each group on day 8. The findings indicated that CD31 enhances neovascularization and increases the number of microvessels, thereby improving the nutritional and oxygen supply to the wound site, which is crucial for wound healing. In addition, Ki67 was significantly upregulated during the wound-healing process, playing a pivotal role in promoting cellular proliferation. CD206⁺ M2-type macrophages inhibited wound inflammation and played a crucial role in tissue repair and regeneration [23]. Based on the results of this study, CD31, Ki-67, and CD206 expression levels in the wound tissue cells of diabetic rats treated with PAA-CaP@AgNps@exo hydrogel were significantly higher than those in other treatment groups (Figure 8a). According to these results, PAA-CaP@AgNps@exo hydrogel may speed the healing of diabetic wounds by promoting angiogenesis and promoting tissue regeneration.

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Figure 8.

PAA-CaP@AgNps@exo hydrogel promotes angiogenesis, cell proliferation, anti-inflammation, re-epithelialization, and collagen deposition. (a) Immunohistochemical staining images of CD31, Ki67, CD206 expression on day 8, and CK10, COLI, COLIII expression on day 16 in the skin wounds of diabetes rats with different treatments; (b) The quantitative results of the CD31, Ki67, CD206, CK10, COLI, and COLIII expression detected by immuno- histochemical staining (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001). BC: Blank control, PC: Positive control, CD31: Platelet endothelial cell adhesion molecule-1, KI67: Marker of Proliferation Ki-67, CD206: Mannose Receptor C-Type 1, CK10: Cytokeratin 10, Col I: Collagen Type I, Col III: Collagen Type III. The black circles represent the experimental results from independent repetitions of the experiment.

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CK10 is integral to the regeneration and repair processes of the epidermal layer of the skin. Furthermore, the differential expression of CK10 across various treatment groups at day 16 was observed to determine the extent of reepithelialization and epithelial differentiation. The results indicated that the PAA-CaP@AgNps@exo hydrogel-treated group exhibited higher CK10 expression and demonstrated a comparatively more mature epithelial structure than the other groups (Figure 8b). The principal collagen types present in the skin are type I collagen (COL I) and type III collagen (COL III). The appropriate deposition of these collagen types can enhance the strength of the skin, facilitate the healing process, and reduce the formation of scars.[24] In this study, the content of these two types of collagen in skin tissue on day 16 of wound healing was investigated using immunohistochemistry. The findings revealed that the group treated with PAA-CaP@AgNps@exo hydrogel exhibited higher COL I and COL III levels than any other group. As a result of these findings, PAA-CaP@AgNps@exo hydrogel is capable of remodeling the extracellular matrix effectively (Figure 8b). These findings indicate that the PAA-CaP@AgNps@exo hydrogel has the potential to expedite the healing of diabetic wounds by enhancing angiogenesis, cell proliferation, anti-inflammatory responses, re-epithelialization, and collagen deposition.

3.2.4. PAA-CaP@AgNps@exo hydrogel accelerates bacteria-infected diabetic wound healing

Wound infection is a serious complication in diabetic wounds, significantly impacting the healing process. The predominant bacterial strains found in infected wounds are S. aureus and E. coli. This study aimed to assess the efficacy of PAA-CaP@AgNps@exo hydrogel in promoting healing in diabetic wounds infected with these bacteria. Surgically created wounds were infected with 1 x 10⁸ cells of S. aureus and E. coli in a 1:1 ratio. On the fourth day, the wounds were cleaned and treated with hydrogels. The established bacteria-infected diabetic wounds rat model is shown in Figure 9(a). The application of PAA-CaP@AgNps@exo hydrogel in treated wounds resulted in significant prevention of infection compared to non-treated infected wounds (Figure 9b,c). Histopathological analysis utilizing HE staining demonstrated enhanced fibroblast proliferation and granulation tissue formation in wounds treated with PAA-CaP@AgNps@exo hydrogels. In addition, the treated wounds exhibited increased collagen deposition, as evidenced by Masson’s trichrome staining, and were characterized by the presence of mature epithelial structures (Figure 9d). The inflammatory cytokines IL-1β and TNF-α at the wound site on day 8 of wound healing were detected using immunofluorescence. It revealed that PAA-CaP@AgNps@exo hydrogel effectively suppressed the presence of inflammatory cytokines IL-1β and TNF-α at the wound site, and it also effectively suppressed the release of inflammatory factors (Figure 9e,f). These results suggest the potential of PAA-CaP@AgNps@exo hydrogel in promoting accelerated healing in infected diabetic wounds.

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Figure 9.

PAA-CaP@AgNps@exo hydrogel accelerates bacteria-infected diabetic wound healing. (a) Schematic diagram of the experimental procedure; (b) Images of wound healing of diabetic rats treated by PAA-CaP@AgNps@exo hydrogel at different time points; (c) Graph of statistical calculations of the residual wound area at different time points (n=3, mean±SD, *p < 0.05, **p < 0.01, ***p < 0.001); The black circles represent the experimental results from independent repetitions of the experiment. (d) Hematoxylin and eosin (HE) and Masson staining images of the skin wounds tissue on day 16; (e) Immunofluorescence staining images of Interleukin-6 (IL-6), and Tumor Necrosis Factor alpha (TNF-alpha), expression at the wound site on day 8; (f) Quantitative results of IL-1, and TNF-, expression in the regenerated skin tissue at day 8 (n = 3, mean ± SD, **p < 0.01, ***p < 0.001). (Hematoxylin and Eosin staining (HE) magnification 50x)

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3.3 Discussion

Diabetic wounds are particularly vulnerable to hyperglycemic conditions, which compromise the viability and functionality of skin cells, thereby impeding their proliferation and migration. This also results in reduced angiogenesis, collectively contributing to delayed wound repair [25]. Furthermore, the presence of elevated glucose levels serves as a nutrient source for bacteria, facilitating biofilm formation and subsequently leading to infections that further hinder the wound healing process [26]. Consequently, it is important to develop effective strategies to expedite wound healing in diabetic patients.

Over the past few years, researchers have been exploring the ability of exosomes derived from various cellular sources to facilitate the healing of challenging skin wounds [27]. It has been demonstrated that exosomes can facilitate the healing of wounds through their ability to regulate inflammatory responses, promote cell proliferation, promote migration, enhance angiogenesis, and inhibit scar formation [28-30]. These findings offer novel insights and strategies for the clinical management of diabetic wounds. UCB is easily collected and is frequently regarded as medical waste. Cord blood plasma contains a variety of biological substances, including proteins, enzymes, and active molecules secreted by cells. The isolation and purification of exosomes from human cord blood plasma results in the production of a substance that contains a multitude of biologically active elements with minimal immunogenicity. This has the potential to be utilized in the treatment of a variety of diseases [31,32]. Currently, little research has been conducted on the potential of UCB exosomes for the healing of diabetic wounds. Moreover, the precise mechanism of action remains uncertain. In this study, cord blood exosomes were initially obtained by centrifugation and were observed to repair the dysfunction of keratinocytes and HUVECs in high-glucose environments. Furthermore, the exosomes were found to increase the proliferation, migration, and tube-forming ability of HUVECs. Sequencing analyses demonstrated that exosomes can influence cell function under high glucose conditions by modulating multiple signaling pathways, including the cell cycle. However, the direct use of exosomes for wound healing therapy has the problems of uncontrolled dosing and short half-life. In recent years, researchers have been practicing to develop effective biocompatible scaffolds as slow-release carriers for exosomes, and one of the more widely used carriers is hydrogel, which has good hydrophilicity, biocompatibility, and porous structure, and it can be effectively loaded with drugs or biologically active substances as a smart delivery system for wounds [33]. Bioprinting techniques for hydrogels also have a prospect in wound healing [34]. Studies have shown that exosomes can be combined with various forms of hydrogels, such as chitosan-silk hydrogel [35], nanofiber hydrogel [36], and natural methylcellulose-chitosan hydrogel [37], all of which have good mechanical properties, water retention, and cytocompatibility, and it can play a more stable and efficient role in the healing effect of exosomes on wounds.

This study draws inspiration from the mineralization of collagen fibers in living organisms. It demonstrates that PAA or other polyelectrolytes can self-assemble with biometallic ions such as Ca²⁺, Cu²⁺, and Zn²⁺ to form bionanomimetic mineralized hydrogels [38,39]. These hydrogels exhibit commendable biocompatibility and biodegradability. Furthermore, metal ions present in hydrogel matrices can interact with protein-based pharmaceuticals, forming aggregates that enhance drug loading while preserving long-term drug efficacy in vivo [40]. Conversely, biomineralized hydrogels can be readily modified by the surrounding reaction environment, enabling ion-responsive degradation and drug release. In the present study, a PAA-CaPs@AgNps@exo nanocomposite hydrogel was synthesized through the use of a PAA self-assembly procedure in conjunction with Ca²⁺ within a phosphate buffer. This process necessitated the utilization of PAA, possessing a carboxyl group (-COOH), as an inherent template. This template subsequently interacted with diffused Ca²⁺ and PO₄^3-, leading to the in situ encapsulation of Ag₃PO₄ nanoparticles and exosomes. The hydrogel can be utilized for the gradual release of exosomes in a solution of physiologically conditioned phosphate. The utilization of this hydrogel in the treatment of diabetic wounds has been demonstrated to facilitate the repair of the skin through the promotion of cell proliferation, collagen deposition, and neovascularization. The utilization of this hydrogel in bacterial-infected diabetic wounds has been demonstrated to exert antimicrobial effects as well as lower inflammatory factors. During this study, a biomimetically mineralized composite hydrogel was utilized to enable the controlled release of exosomes and nanosilver particles within the gel under physiological phosphate conditions. These results indicate that this approach enhanced wound healing through a variety of mechanisms, offering an innovative approach to diabetic wound dressings.